Does anyone know of a good, reliable way to produce images of negatively
stained specimens after immunogold labeling? I have tried the following
two methods with bacterophages.
Method A. The protocol followed was:
(1) Adsorb properly diluted phage sample onto glow-discharge plastic-carbon
coated grid for one minute.
(2) Incubate with 1st antibody for 30 minutes.
(3) Rinse the grids with buffer, or pass the grids over drops of buffer.
(4) Incubate with 2nd antibody-gold for 30 minutes.
(5) Rinse again.
(6) Negative stain.
The difficulty with this method I have encountered is that after the
procedure is applied either the film breaks as soon as the electron beam
hits, or most of the phages are lost. (Without IEM, the negative-stained
phages were fine).
Method B: Incubate the phages with 1st antibody and 2nd antibody-gold in a
microfuge tube and then negative stain. This method works well, but the
specimen appears to have a very high background of gold particles.
I wonder if others have similar difficulties and how they are dealing with
them. Any information or suggestions would be appreciated.
Eleana Sphicas
psphicas@pipeline.com
Go to Gold Background on Formvar or Gold Probe Storage or Gold Labeling for LM
In general I have always had success with your method #1. I would suggest a
Blocking step after applying the phage and before applying the primary antibody.
Also use as small a mesh size for your grid as possible and use nickel not
copper. I have always done it with 400 mesh grids, but smaller are
available if you a re have trouble with the film breaking. Other wise just
try to minimize mechanical damage as much as possible.
Good luck
Greg Erdos
E-mail: gwe@biotech.ufl.edu
I have had sucess in the past with a slight variation of method B:
My antibody-labeled sample was incubated with Protein A-gold for 60 min
at 20°C, and then separated from the unbound labed by wasing (four cycles)
in phosphate-buffered saline, employing a Beckman airfuge centrifugation
conditions of 134,000g with rotor 95A or at 196,000g in the 110A rotor.
The final pellet was taken up in 10µl of buffer and then negatively
stained for electron microscopy.
Ref: J. of Struct. Biology, 106: 221-236 (1991)
Good Luck,
Margaret(Peggy) Bisher
e-mail: peggy@research.nj.nec.com
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